Project Summary Kidney transplantation is life-saving for people with end stage kidney disease (ESKD). However, complications including opportunistic infection and chronic T cell- and antibody-mediated rejection reduce long-term graft survival. Seropositivity for cytomegalovirus (CMV) is associated with higher rates of chronic rejection after transplantation. Chronic exposure to CMV induces immunosenescence in CMV-responsive memory T cells, including loss of proliferative capacity and differentiation potential, with maintained production of inflammatory cytokines. In consequence, these cells become less protective, and may even promote rejection. My preliminary data suggest that the development of senescence in response to CMV is accelerated after transplantation. In addition, T cell mediated infiltration of inflammatory macrophages into kidney grafts can induce rejection. Macrophages are a major site of CMV infection. Together, these observations led to the hypothesis that senescent CMV-responsive T cells contribute to chronic rejection that will be tested through two aims. The goal of the first aim is to characterize senescence in CMV-responsive T cells post- transplant. CMV-responsive T cells will be analyzed at post-transplant time points before and after the onset of acceleration of senescence. Characterization will include in-depth gene expression profiling by single cell RNA sequencing and functional assays for chromatin accessibility, telomere length, and proliferation. The goal of the second aim is to determine whether CMV-responsive T cells and inflammatory macrophages influence susceptibility to chronic rejection. Quantity and localization of infiltration of both CMV-specific T cells and macrophages into kidney allografts will be measured in archived biopsy specimens, comparing patients diagnosed with chronic rejection to a control group of patients with chronicity. Phenotypic analysis of blood T cells at the time of transplant biopsy will identify populations correlated with rejection, which in the long-term may provide a new diagnostic of rejection. The career plan is for the PI to become an independent investigator researching the effects of immune complications on kidney transplant. The research environment at Stanford is well suited for these studies, given access to core facilities for sequencing, flow cytometry, and microscopy. The aims provide opportunities to gain expertise in collaboration with bioinformaticians, statisticians, nephrologists, and pathologists, which are key to the career plan. Specifically, Aim 1 involves collaboration with Dr. Purvesh Khatri for complex data analysis of sequencing data sets. Aim 2 involves collaboration with transplant nephrologists Drs Jane Tan and Paul Grimm and pathologist Dr. Neeraja Kambham for analysis and interpretation of biopsy results. It also involves collaboration with renal cell carcinoma researcher Dr. Wendy Fantl and statistician Dr. Robert Tibshirani for highly multiplexed analysis of biopsy specimens. These aims are formulated to gain understanding of the role of CMV-responsive T cells in chronic rejection, potentially to produce new therapies to chronic rejection, and to support the applicant?s transition to independence.